Combining CRISPR activation and interference capabilities using dCas9 and G-quadruplex structures.

Area of Science:

• Molecular Biology
• Gene Regulation
• Biophysics

Background:

• The c-Myc oncogene is a critical regulator of cell proliferation, and its dysregulation is implicated in various cancers, including Burkitt's Lymphoma.
• G-quadruplex (GQ) structures in promoter regions can influence gene transcription.
• CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) are powerful tools for gene regulation.

Purpose of the Study:

• To investigate the efficacy of targeting a G-quadruplex-forming sequence (PQS) in the c-Myc promoter using nuclease-dead Cas9 (dCas9) for gene regulation.
• To achieve CRISPR-mediated transcriptional suppression and activation of c-Myc at both RNA and protein levels.
• To elucidate the mechanistic details of CRISPR-dCas9 interaction with the c-Myc promoter and its impact on transcription.

Main Methods:

• Utilized CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems with dCas9 to target a PQS in the c-Myc promoter.
• Conducted experiments in a Burkitt's Lymphoma cell line and in vitro.
• Employed quantitative real-time PCR (qRT-PCR) for mRNA analysis, Western blotting for protein level assessment, and cell viability assays.
• Performed extensive in vitro biophysical studies to analyze molecular interactions.

Main Results:

• Targeting the template strand near the PQS with dCas9 destabilized the GQ, leading to a significant increase in c-Myc mRNA (2.1-fold) and protein (1.6-fold) levels.
• Targeting individual sites on the non-template strand (NTS) with dCas9 reduced c-Myc mRNA (1.8-fold) and protein (2.5-fold) levels.
• Simultaneous targeting of two NTS sites resulted in substantial suppression: 3.6-fold for mRNA and 9.8-fold for protein.
• Cell viability assays showed corresponding reductions (1.7-fold and 4.7-fold) with single and dual NTS targeting, respectively.
• In vitro biophysical studies quantitatively supported the cellular findings and provided mechanistic insights.

Conclusions:

• CRISPR-dCas9 targeting of the c-Myc promoter, particularly near a PQS, is an effective strategy for both gene activation and repression.
• The modulation of GQ stability by dCas9 plays a crucial role in regulating c-Myc transcription.
• This approach offers a potent and versatile method for controlling oncogene expression, with potential therapeutic implications.